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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Time (min.)
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Figure 2: LC-MS/MS chromatogram of JWH metabolites found in an authentic urine
sample.
Figure 1: LC-MS/MS chromatogram of a 1 ng/mL JWH metabolites calibration standard.
References
[1] T. Sobolevsky, I. Prasolov, G. Rodchenkov, Detection of JWH-018 Metabolites in Smoking Mixture
Post-Administration Urine, Forensic Sci. Int., 200 (2010) 141.
[2] A. Grigoryev, S. Savchuk, A. Melnik, N. Moskaleva, J. Dzhurko, M. Ershov, A. Nosyrev, A. Vedenin, B. Izotov, I.
Zabirova, V. Rozhanets. Chromatography–Mass Spectrometry Studies on the Metabolism of Synthetic Cannabinoids
JWH-018 and JWH-073, Psychoactive Components of Smoking Mixtures, J. Chromatogr. B, 879 (2011) 1126.
[3] B. Logan, S. Kacinko, M. McMullin, A. Xu, R. Middleberg, Technical Bulletin: Identification of Primary JWH-018 and
JWH-073 Metabolites in Human Urine, (2011).
Ultra Biphenyl Columns (USP L11)
Physical Characteristics:
particle size: 3µm or 5µm, spherical endcap: fully endcapped
pore size: 100Å pH range: 2.5 to 8
carbon load: 15% temperature limit: 80°C
Description cat.#
5µm Columns
50mm, 2.1mm ID 9109552
50mm, 2.1mm ID
(with Trident Inlet Fitting) 9109552-700
Resprep® SPE Cartridges
(Bonded Reversed Phases)
Hydrophobic (nonpolar) silica-based adsorbents, used to extract
hydrophobic analytes from polar matrices, such as water (e.g.,
pesticides from water).
6mL/500mg
C18 (high load, endcapped) 24052
0.5 0.0 1.0
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1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Time (min.)
0
LC_CF0530
Column:Ultra Biphenyl (cat.# 9109552); Dimensions: 50 mm x 2.1 mm ID; Particle Size: 5 µm; Pore Size: 100 Å; Temp.: 25 °C; Sample: Diluent:
50:50 mobile phase; Conc.: 1 ng/mL extracted spiked sample; Inj. Vol.: 10 µL; Mobile Phase:A: water + 0.05% acetic acid (pH approx. 3.4),
B: acetonitrile + 0.05% acetic acid;Flow: 0.5mL/min.; Gradient (%B): 0 min. (45%), 2.00 min. (45%), 6.00 min. (85%), 6.10 min. (95%),
7.00 min. (95%), 7.10 min. (45%), 8.50 min. (stop); Detector: API 4000; Model #: API 4000; Ion Source: TurboIonSpray®; Ion Mode: ESI+; Ion
Spray; Mode: MRM; Instrument:API LC MS-MS; For complete conditions and transitions, visit and enter LC_CF0530 in the
search.
Peaks RT (min.)
1. JWH-073 4-hydroxybutyl 2.04
2. JWH-073 N-butanoic acid 2.13
3. JWH-018 N-pentanoic acid 2.59
4. JWH-018 5-hydroxypentyl 2.57
5. JWH-073 6-hydroxyindole 3.52
6. JWH-073 5-hydroxyindole 3.68
7. JWH-073 7-hydroxyindole 3.95
8. JWH-018 6-hydroxyindole 4.00
9. JWH-018 5-hydroxyindole 4.13
10. JWH-018 7-hydroxyindole 4.34
11. JWH-073 4-hydroxyindole 5.15
12. JWH-018 4-hydroxyindole 5.44
LC_CF0533
Peaks RT (min.)
1. Suspected unknown metabolite 1.90
2. JWH-073 N-butanoic acid 2.13
3. JWH-018 N-pentanoic acid 2.59
4. JWH-018 5-hydroxypentyl + unknown metabolite 2.55
5. JWH-018 6-hydroxyindole 3.99
the same transitions as JWH-018 5-hydroxy-
pentyl. This peak was not present in any
of the blank samples and, based on recent
work by NMS Labs, is thought to be JWH-018
4-hydroxypentyl [3].
Although JWH-073 n-butanoic acid was
present in several samples, no JWH-073
4-hydroxybutyl was found. However, a large
peak with the same transitions as JWH-073
4-hydroxybutyl was detected at a slightly
earlier retention time compared to the
JWH-073 4-hydroxybutyl metabolite. Post-
extraction spiking experiments confirmed
that the observed peak was not due to
JWH-073 4-hydroxybutyl. The unknown peak
was not observed in any blank samples, sug-
gesting that it is also an unknown metabolite
of either JWH-018 or JWH-073. Comparison
to an NMS Labs report indicates this peak is
most likely JWH-073 3-hydroxybutyl [3].
Summary
The extraction and chromatographic meth-
ods shown here perform well for the analysis
of JWH-018 and JWH-073 metabolites in
urine. The mid-range pH SPE extraction
allows both mono-hydroxylated and car-
boxylated metabolites to be recovered from
a single extraction. In addition, the Ultra
Biphenyl column provides enough retention
for the hydrophilic carboxylated metabolites,
as well as the selectivity needed to separate
positional isomers of the mono-hydroxylated
metabolites.
For the complete version of this technical
article, visit
Sample was prepared according to the following method:
1) Spike 1 mL blank urine sample with analytes and internal
standards.
2) Hydrolyze sample:
- Add 1 mL solution of beta-glucuronidase from keyhole limpet
(Sigma-Aldrich cat.# G8132). Solution is prepared at a concentra-
tion of 5,000 Fishman units/mL in 100 mM ammonium acetate
buffer (pH = 5.0).
- Incubate at 60 °C for 3 hours.
3) Extract sample on 6 mL, 500 mg C18 high-load endcapped
Resprep® SPE cartridge (cat.# 24052):
- Add 1 mL 5 mM ammonium acetate + 0.1% acetic acid (pH = 4.2)
to sample.
- Condition cartridge with 3x 1 mL acetonitrile.
- Condition cartridge with 3x 1 mL 5 mM ammonium acetate
+ 0.1% acetic acid.
- Apply sample and allow to pass through under gravity.
- Rinse with 3x 1 mL 5 mM ammonium acetate + 0.1% acetic acid.
- Dry cartridge with vacuum for 10 minutes.
- Elute with 3 mL acetonitrile followed by 3 mL butyl chloride.
4) Concentrate sample:
- Evaporate sample to dryness under nitrogen at 40 °C.
- Reconstitute in 0.5 mL water + 0.05% acetic acid:acetonitrile
+ 0.05% acetic acid (50:50).
Acknowledgement:Special thanks to Cayman Chemical for
reference standards
(See Figure 1 for instrument conditions and extraction procedure.)
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